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Treatment options are lacking to prevent photoreceptor death and subsequent vision loss. Previously, we demonstrated that reprogramming metabolism via the pharmacologic activation of PKM2 is a novel photoreceptor neuroprotective strategy. However, the features of the tool compound used in those studies, ML-265, preclude its advancement as an intraocular, clinical candidate. This study sought to develop the next generation of small-molecule PKM2 activators, aimed specifically for delivery into the eye. Compounds were developed that replaced the thienopyrrolopyridazinone core of ML-265 and modified the aniline and methyl sulfoxide functional groups. Compound 2 demonstrated that structural changes to the ML-265 scaffold are tolerated from a potency and efficacy standpoint, allow for a similar binding mode to the target, and circumvent apoptosis in models of outer retinal stress. To overcome the low solubility and problematic functional groups of ML-265, compound 2’s efficacious and versatile core structure for the incorporation of diverse functional groups was then utilized to develop novel PKM2 activators with improved solubility, lack of structural alerts, and retained potency. No other molecules are in the pharmaceutical pipeline for the metabolic reprogramming of photoreceptors. Thus, this study is the first to cultivate the next generation of novel, structurally diverse, small-molecule PKM2 activators for delivery into the eye. Full article

The vacuolar H⁺-adenosine triphosphatase (ATPase) subunit V0C (ATP6V0C), a proton-conducting, pore-forming subunit of vacuolar ATPase, maintains pH homeostasis and induces organelle acidification. The intracellular and extracellular pH of cancer cells affects their growth; however, the role of ATP6V0C in highly invasive esophageal cancer cells (ECCs) remains unclear. In this study, we examined the role of ATP6V0C in glucose metabolism in ECCs. The ATP6V0C depletion attenuated ECC proliferation, invasion, and suppressed glucose metabolism, as indicated by reduced glucose uptake and decreased lactate and adenosine triphosphate (ATP) production in cells. Consistent with this, expression of glycolytic enzyme and the extracellular acidification rate (ECAR) were also decreased by ATP6V0C knockdown. Mechanistically, ATP6V0C interacted with pyruvate kinase isoform M2 (PKM2), a key regulator of glycolysis in ECCs. The ATP6V0C depletion reduced PKM2 phosphorylation at tyrosine residue 105 (Tyr¹⁰⁵), leading to inhibition of nuclear translocation of PKM2. In addition, ATP6V0C was recruited at hypoxia response element (HRE) sites in the lactate dehydrogenase A (LDHA) gene for glycolysis. Thus, our data suggest that ATP6V0C enhances aerobic glycolysis and motility in ECCs. Full article

Mitochondria evolved from free-living bacteria via endocytosis within eukaryotic host cells millions of year ago. We hypothesized that antibiotics cause mammalian mitochondrial damage while causing bacterial lethality. Mitochondrial toxicity of azithromycin in human mammary epithelia MCF-12A and fibroblasts were tested by fluorescent and transmission electron microscopy. Gene expression and DNA damage were tested by real-time polymerase chain reaction (qPCR) and ELISA. We found azithromycin suppressed the mitochondrial membrane potential gradient of MCF-12A cells and fibroblasts. Ultrastructure exams showed that the antibiotic caused vacuolated and swollen mitochondria with disrupted cristae in MCF-12A cells and fibroblasts compared to the morphology of mitochondria in the cells without antibiotic treatment. Fluorescent microscopy also showed azithromycin-induced mitochondrial reactive oxygen species (ROS), superoxide, after 3 h of culture. The DNA oxidative damage product, 8-hydroxy-2’-deoxyguanosine (8-OHdG, significantly increased in the media after MCF-12A cells and fibroblasts were cultured in the media containing azithromycin for 24 h. Azithromycin upregulated gene expression of hypoxia inducible factor 1 alpha (HIF1a), glycolytic enzymes including hexokinase 2 (HK2), phosphofructokinase 1 (PFKM), pyruvate kinase muscle isozyme M2 (PKM2), and glucose transporters in MCF-12A cells and fibroblasts. Lactate production also increased in the culture media. After treatment with azithromycin, healthy MCF-12A and fibroblast cells increased aerobic glycolysis—the “Warburg Effect”—to generate energy. In summary, azithromycin caused mitochondrial toxicity, ROS overproduction, DNA oxidative damage, upregulation of the HIF1a gene, and aerobic glycolysis in healthy mammalian cells. Over-usage of antibiotics could contribute to tumorigenesis and neurodegeneration and aggravate existing mitochondria-associated diseases. Full article